Summary of Work: Summary of Work: Exogenous DNA damaging agents such as cisplatinin, nitrogen mustard, and psoralen create interstrand DNA crosslinks. Such non-coding lesions must be repaired to ensure accurate replication of the genome and viability of the organism. The Drosophila mus308 mutation, which confers hypersensitivity to nitrogen mustard but not the monofunctional agent methyl-methane sulfonate, identified a DNA polymerase likely involved in processing DNA crosslinks. Based on homology to the Drosophila mus308 gene and another Family A DNA polymerase, we previously cloned part of the cDNA for human DNA polymerase theta. The human cDNA encoded a putative DNA polymerase of 1762 amino acids with a calculated molecular weight of ~200 kDa. We have overproduced a 100 kDa and 200 kDa protein in E. coli and baculoviral infected cells and purified the recombinant protein. Both polypeptides have the expected DNA polymerase activity. Site-directed alterations of active site residues in the DNA polymerase have been produced to confirm the DNA polymerase function. Further analysis of the gene indicates a much larger coding region for pol theta, encoding a protein with a molecular weight of 300 kDa, containing motifs for DNA polymerase, exonuclease, and a helicase. We recently isolated the entire cDNA for the pol theta encoding a protein of 298 kDa. This full length cDNA has been overexpressed in insect cells by a recombinant baculovirus and protein purified. Current efforts are focused to improve the purification and yield of protei